The success of gene therapy techniques depends on the ability to achieve sufficient expression of transferred genes in a manner safe to humans. Retroviruses are often used as a delivery system (otherwise expressed as a delivery vehicle or delivery vector) for inter alia the transfer of a nucleotide of interest (NOI), or a plurality of NOIs, to one or more sites of interest. There has also been considerable interest in the development of lentiviral vector systems because lentiviruses are able to infect non-dividing cells (Lewis & Emerman (1993) J. Virol. 68:510). In addition lentiviral vectors enable very stable long-term expression of the gene of interest. This has been shown to be at least one year for transduced rat neuronal cells in vivo (Bienemann et al. (2003) Mol. Ther. 5:588).
The vector purification process is an important step in clinical gene transfer therapies, and is directly linked to safety and efficacy in terms of purity and titre. In most small-scale experimental applications, vectors can be concentrated and purified by relatively simple methods using centrifugation techniques. However, scaling up the purification methods for large-scale production for clinical use represents a major challenge. In particular, when considering production of vector for human use, additional steps such as sterilisation by filtration must be taken to ensure the purity and safety of the vector preparation.
Vector preparation methods generally comprise obtaining vector from cells making vector either stably or transiently and purifying the viral vector using, for example, chromatography. In industrial processes, the final step is the sterilisation step and at least one ultrafiltration step is usually used prior to the sterilisation step so as to concentrate the viral vector and/or to exchange the buffer in which the viral vector is kept.
Several publications describe the purification of viruses from cells, mostly discussing the use of specific chromatographic matrices for purification of the virus from a cell lysate. For example, U.S. Pat. No. 6,261,823 and U.S. Pat. No. 5,661,023 disclose methods encompassing the use of anion exchange resin alone while U.S. Pat. No. 5,837,520 discloses a sequential combination of anion exchange followed by affinity chromatography. Yamada et al. shows that lentivirus vector purification using anion exchange HPLC leads to improved gene transfer (Yamada et al. (2003) Biotechniques 34(5):1074-8, 1080).
There remains a need for a large-scale retroviral vector purification method that yields pure product while preserving high titres. The present invention addresses this need.